Lung cancers are the most common malignancies and are the leading cause of cancer-related deaths worldwide. Immunotherapies are transforming treatment regimes, and trials conducted in lung cancer have already demonstrated improvements in long-term survival outcomes. However, existing therapies are not effective in all patients, stressing a need to better characterize the complex molecular and immunological processes associated with treatment responsiveness and disease progression. Although T cells have been the primary target of immunotherapies in lung cancer, there is a growing appreciation that T cell-mediated anti-tumor immunity can be influenced by actions of other immune cells within the tumor microenvironment (TME). Tumor-infiltrating (TI) B cells have been shown to have dual roles in tumor progression generally, but in non-small cell lung cancers (NSCLC), specifically adenocarcinomas (LUAD), particular B cell subsets have been shown to both promote T cell anti-tumor responses and positively impact long-term survival outcomes. Indications of a role for specific antibody (Ab) responses in LUAD have also recently been reported, including evidence of focused tumor antigen-specific responses. However, characterizations of anti-tumor B cells and their functional signatures have been limited by the study of only a small number of pre-selected surface markers in relatively small numbers of patients. Nonetheless, it is evident that B cell-mediated roles in LUAD tumor progression are variable among patients, and indicates that a more comprehensive and integrated characterization of TI B cell phenotypic variation and the associated effects on the anti-tumor T cell response has great potential to inform our understanding of disease progression, as well as the development of novel, potentially personalized, therapies. The overall goal of this proposal is to define TI B cell signatures that are associated with the T cell-mediated anti-tumor response in human lung cancer. To achieve this goal, we will leverage several cutting-edge techniques to first comprehensively profile lymphocytes (B and T cells) in tumor and paired normal tissues from a large cohort of early-stage LUAD patients, including the first pairing of this type of data with Ab repertoire sequencing as a means to interrogate critical functional Ab response signatures in LUAD B cells. Importantly, the single-cell profiling approach proposed will leverage B cell markers already determined to be associated with long-term LUAD survival outcomes, so that distinct functional B cell profiles and their associations with TI T cells can be identified specifically in the context of disease progression (Aim 1). These large-scale statistical associations will be validated and then serve as a framework for conducting functional in vitro culture and serum- based analyses to assess whether prognostically favorable B cell subsets identified have distinct functional phenotypes, associate with tumor antigen-specific Ab responses, and have differential effects on T cell activation and effector function (Aim 2). Successful completion of this project will facilitate novel discovery and biomarkers critical for the development of novel therapeutics in human lung cancer.